Alpha-amylases and polynucleotides encoding same

ABSTRACT

The present disclosure relates to isolated polypeptides having alpha-amylase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing the polypeptides, and method of using polypeptides, including in ethanol production processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/653,154filed Jun. 17, 2015, now U.S. Pat. No. 9,765,317, which is a 35 U.S.C.371 national application of PCT/US2013/074957 filed Dec. 13, 2013 whichclaims priority or the benefit under 35 U.S.C. 119 of U.S. provisionalapplication No. 61/738,145 filed Dec. 17, 2012 the contents of which arefully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to alpha-amylases, polynucleotidesencoding the alpha-amylases, methods of producing the alpha-amylases,and methods of using the alpha-amylases. In embodiments of the presentdisclosure, raw starch degrading activity is improved.

Description of the Related Art

Enzymatic degradation of starch is part of many industrial processesincluding brewing, production of glucose or high fructose syrups andproduction of drinking or fuel ethanol. In its natural state, starch isquite resistant against degradation by many enzymes, and thereforeindustrial enzymatic degradation of starch is traditionally initiated bya heating step where starch is gelatinized, which renders the starchmore sensitive to many enzymes. Some enzymes are able to act onungelatinized starch, and are commonly referred to as having raw starchdegrading activity. The use of these enzymes permits for improvedprocesses, including, for example, reducing the heating step inprocessing starch.

Alpha-amylases (alpha-1,4-glucan 4 glucanohydrolases, EC. 3.2.1.1)constitute a group of enzymes which catalyze hydrolysis of starch andother linear and branched 1,4 glucosidic oligo and polysaccharides.Alpha-amylase enzymes have been used for a variety of differentindustrial purposes, including starch liquefaction, ethanol production,textile desizing, textile washing, starch modification in the paper andpulp industry, brewing, and baking.

WO 2010/091221 discloses a polypeptide having alpha-amylase activityfrom Aspergillus terreus. Database UniProt XP002576027 discloses thenucleic acid sequence from the Q0C881 (Aspergillus terreus) genome foran alpha-amylase.

WO 2008/080093 discloses alpha-amylases and glucoamylases and their usein making biofuel.

There remains a need in the art for improved alpha-amylases, includingalpha-amylases that have raw starch degrading activity.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides having alpha amylaseactivity selected from the group consisting of:

(a) a polypeptide comprising or consisting of an amino acid sequence ofthe mature polypeptide of the amino acid sequence of SEQ ID NO: 2, SEQID NO:4 or SEQ ID NO:6;

(b) a polypeptide comprising an amino acid sequence having at least 80%sequence identity to the mature polypeptide of the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO:6;

(c) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions with the mature polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5. In embodiments,polypeptides of the present disclosure are isolated.

The present invention also relates to polynucleotides encoding thepolypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention further relates to a transgenic plant, plant partor plant cell transformed with a polynucleotide encoding a polypeptideof the present invention.

In yet further aspects, the present invention relates to compositionscomprising a polypeptide of the present invention, includingcompositions for producing ethanol.

The present invention also relates to method for the production ofethanol using a polypeptide of the present invention. The presentinvention also relates to method for the production of ethanol fromungelatinized starch using a polypeptide of the present invention.

Definitions

Alpha-amylase activity: The term “alpha-amylase activity” is definedherein as an activity that catalyzes the endohydrolysis of(1,4)-alpha-D-glucosidic linkages in polysaccharides containing three ormore (1,4)-alpha-linked D-glucose units. The term “alpha-amylaseactivity” corresponds to the enzymes grouped in E.C. 3.2.1.1. Forpurposes of the present invention, alpha-amylase activity is determinedaccording to the procedure described in the “Example” section.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide of the presentinvention having one or more (e.g., several) amino acids absent from theamino and/or carboxyl terminus of a mature polypeptide or domain,wherein the fragment has alpha-amylase activity. In one aspect, afragment contains at least 497 amino acid residues, at least 526 aminoacid residues, or at least 555 amino acid residues of SEQ ID NOS: 2 or6. In another aspect, a fragment contains at least 528 amino acidresidues, at least 559 amino acid residues, or at least 590 amino acidresidues of SEQ ID NO:4.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample. Forexample, the polypeptide of the present invention may be used inindustrial applications in the form of a fermentation broth product,that is, the polypeptide of the present invention is a component of afermentation broth used as a product in industrial applications (e.g.,ethanol production). The fermentation broth product will in addition tothe polypeptide of the present invention comprise additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. The fermentation broth may optionally be subject to one ormore purification (including filtration) steps to remove or reduce onemore components of a fermentation process. Accordingly, an “isolated”polypeptide of the present invention may be present in such afermentation broth product.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. It is also known in the art thatdifferent host cells process polypeptides differently, and thus, onehost cell expressing a polynucleotide may produce a different maturepolypeptide (e.g., having a different C-terminal and/or N-terminalamino) as compared to another host cell expressing the samepolynucleotide.

In one aspect, the mature polypeptide is amino acids 1 to 585 of SEQ IDNO: 2 based on the SignalP (Nielsen et al., 1997, Protein Engineering10: 1-6) that predicts amino acids −1 to −21 of SEQ ID NO: 2 are asignal peptide.

In another aspect, the mature polypeptide is amino acids 1 to 622 of SEQID NO: 4 based on the SignalP (Nielsen et al., 1997, Protein Engineering10: 1-6) that predicts amino acids −1 to −21 of SEQ ID NO: 4 are asignal peptide.

In another aspect, the mature polypeptide is amino acids 1 to 607 of SEQID NO: 6 based on the SignalP (Nielsen et al., 1997, Protein Engineering10: 1-6) that predicts amino acids −1 to −21 of SEQ ID NO: 6 are asignal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving alpha-amylase activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having alpha-amylase activity.

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Alpha-Amylase Activity

In embodiments, the present disclosure relates to new alpha-amylasesequences. The new alpha-amylase sequences include the maturepolypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO:4 andthe mature polypeptide of SEQ ID NO:6. A mature polypeptide of SEQ IDNO:2 is also shown as the amino acid sequence of SEQ ID NO: 1 (residues1-585). A mature polypeptide of SEQ ID NO:4 is also shown as the aminoacid sequence of SEQ ID NO: 3 (residues 1-622). A mature polypeptide ofSEQ ID NO:6 is shown as the amino acid sequence of SEQ ID NO: 5(residues 1-607).

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have alpha-amylase activity. Inone aspect, the polypeptides differ from the mature polypeptide of SEQID NO: 2 by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acids.

In another aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. A polypeptide of the present inventionpreferably comprises or consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or is a fragment thereof havingalpha-amylase activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO:4 ofat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have alpha-amylase activity. Inone aspect, the polypeptides differ from the mature polypeptide of SEQID NO:4 by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or9 amino acids.

In another aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 4. A polypeptide of the present inventionpreferably comprises or consists of the amino acid sequence of SEQ IDNO: 4 or an allelic variant thereof; or is a fragment thereof havingalpha-amylase activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the polypeptide of SEQ ID NO:6 of at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have alpha-amylase activity. In oneaspect, the polypeptides differ from the mature polypeptide of SEQ IDNO:6 by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acids.

In another aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 6. A polypeptide of the present inventionpreferably comprises or consists of the amino acid sequence of SEQ IDNO: 6 or an allelic variant thereof; or is a fragment thereof havingalpha-amylase activity.

The present invention relates to an isolated polypeptide havingalpha-amylase activity encoded by a polynucleotide that hybridizesmedium stringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with a nucleicacid sequence encoding the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO:3, or SEQ ID NO:5, or (ii) the full-length complementof the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:3,or SEQ ID NO:5.

In another embodiment, the present invention relates to an isolatedpolypeptide having alpha-amylase activity encoded by a polynucleotidehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5 of at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions. In an embodiment, the number of amino acidsubstitutions, deletions and/or insertions introduced into the maturepolypeptide is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 aminoacids.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein. Examples of conservativesubstitutions are within the groups of basic amino acids (arginine,lysine and histidine), acidic amino acids (glutamic acid and asparticacid), polar amino acids (glutamine and asparagine), hydrophobic aminoacids (leucine, isoleucine and valine), aromatic amino acids(phenylalanine, tryptophan and tyrosine), and small amino acids(glycine, alanine, serine, threonine and methionine). Amino acidsubstitutions that do not generally alter specific activity are known inthe art and are described, for example, by H. Neurath and R. L. Hill,1979, In, The Proteins, Academic Press, New York. Common substitutionsare Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Variants of the polypeptides may be constructed on the basis of thepolynucleotide presented as the mature polypeptide coding sequence,e.g., a subsequence thereof, and/or by introduction of nucleotidesubstitutions that do not result in a change in the amino acid sequenceof the polypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-amylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, as described herein. In oneaspect, the present invention relates to polynucleotides that hybridizesunder medium stringency conditions, medium-high stringency conditions,high stringency conditions, or very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 orSEQ ID NO: 5, or (ii) the full-length complement of the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:5.

In another embodiment, the present invention relates to an apolynucleotide having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are Aspergillus nidulans orAspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicusbar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The host cell may be anycell useful in the recombinant production of a polypeptide of thepresent invention, e.g., a prokaryote (bacterial cell) or a eukaryote(such as a mammalian, insect, plant, or fungal cell).

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota as well as the Oomycota and all mitosporic fungi (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: a) cultivating a host cellcomprising the polynucleotide encoding the polypeptide of the presentinvention operably linked to one or more control sequences that directthe production of the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

In a preferred aspect, the cell is an Aspergillus cell, such as,Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillusterreus.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Such polypeptide compositions maybe prepared in accordance with methods known in the art and may be inthe form of a liquid or a dry composition. The polypeptide to beincluded in the composition may be stabilized in accordance with methodsknown in the art.

The polypeptide composition may be in the form of granulate, a microgranulate or a powder. Methods of preparing such compositions are wellknown in the art.

The polypeptide composition may be in the form of a fermentation brothproduct. The fermentation broth product will in addition to thepolypeptide of the present invention comprise additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. The fermentation broth may optionally be subject to one ormore purification (including filtration) steps to remove or reduce onemore components of a fermentation process.

The polypeptide composition may further comprise an enzyme selected fromthe group comprising of another alpha-amylase (EC 3.2.1.1), abeta-amylase (E.C. 3.2.1.2), a glucoamylase (E.C. 3.2.1.3), apullulanases (E.C. 3.2.1.41), a phytase (E.C. 3.1.2.28) and a protease(E.C. 3.4.), and combinations thereof (e.g., the polypeptide of thepresent invention and a glucoamylase. or the polypeptide of the presentinvention and a glucoamylase and a protease).

In a particular aspect, the polypeptide composition further comprises aglucoamylase. The polypeptide may be combined with commercialglucoamylase, such as, the glucoamylase preparation supplied byNovozymes A/S as SPIRIZYME FUEL. The glucoamylase may also be derivedfrom a strain of Aspergillus sp., such as Aspergillus niger, or from astrain of Talaromyces sp. and in particular derived from Talaromycesleycettanus such as the glucoamylase disclosed in U.S. Pat. No. Re.32,153, Talaromyces duponti and/or Talaromyces thermopiles such as theglucoamylases disclosed in U.S. Pat. No. 4,587,215 and more preferablyderived from Talaromyces emersonii. In one aspect, the glucoamylase isderived from Talaromyces emersonii strain CBS 793.97 and/or having thesequence disclosed as SEQ ID NO: 7 in WO 99/28448. In another aspect,the glucoamylase activity is derived from a strain of the genusTrametes, preferably Trametes cingulata. Further glucoamylases includethe glucoamylase having the amino acid sequence of the maturepolypeptide of SEQ ID NO: 2 in WO 2006/069289. Glucoamylase may alsoinclude glucoamylases from the genus Pachykytospora, preferablyPachykytospora papyracea or the E. coli strain deposited at DSMZ andgiven the no. DSM 17105, and including the glucoamylase having the aminoacid sequence of the mature polypeptide of mature polypeptide of SEQ IDNO: 5 in WO 2006/069289. Further glucoamylases include those which havean amino acid sequence having at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or even at least 99% homology to the aforementioned aminoacid sequence.

The dosage of the polypeptide composition of the invention and otherconditions under which the composition is used may be determined on thebasis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having alpha-amylase activity, or compositions thereof.

The polypeptide or the composition of the present invention may be usedin starch conversion, starch to sugar conversion and ethanol productionetc, e.g., in liquefying and/or saccharifying a gelatinized starch, agranular starch, or a partly gelatinized starch. A partly gelatinizedstarch is a starch which to some extent is gelatinized, i.e., whereinpart of the starch has irreversibly swelled and gelatinized and part ofthe starch is still present in a granular state.

The polypeptide or the composition of the present invention may be usedin a process for liquefying a gelatinized starch, a granular starch, ora partly gelatinized starch substrate in aqueous medium with thepolypeptide of the present invention.

A preferred use of a polypeptide of the present invention is in afermentation process to produce glucose and/or maltose suitable forconversion into a fermentation product by a fermenting organism,preferably a yeast. Such fermentation processes include a process forproducing ethanol for fuel or drinking ethanol (portable alcohol), aprocess for producing a beverage, a process for producing desiredorganic compounds, such as citric acid, itaconic acid, lactic acid,gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate,glucono delta lactone, or sodium erythorbate; ketones; amino acids, suchas glutamic acid (sodium monoglutaminate), but also more complexcompounds such as antibiotics, such as penicillin, tetracyclin; enzymes;vitamins, such as riboflavin, B12, beta-carotene; hormones, which aredifficult to produce synthetically.

In a preferred embodiment, the polypeptide of the present invention isused in a process comprising fermentation to produce a fermentationproduct (e.g., ethanol), from a gelatinized starch. Such a process forproducing ethanol from gelatinized starch by fermentation comprises: (i)liquefying the gelatinized starch with a polypeptide with alpha-amylaseactivity of the present invention; (ii) saccharifying the liquefied mashobtained; (iii) fermenting the material obtained in step (ii) in thepresence of a fermenting organism. Optionally the process furthercomprises recovery of the ethanol. The saccharification and fermentationmay be carried out as a simultaneous saccharification and fermentationprocess (SSF process).

In another preferred embodiment, the polypeptide of the presentinvention is used in a process comprising fermentation to produce afermentation product, e.g., ethanol, from an ungelatinized (“raw”)starch. Such a process for producing ethanol from ungelatinizedstarch-containing material by fermentation comprises: (i) contacting theungelatinized starch with a polypeptide with alpha-amylase activity ofthe present invention to degrade the ungelatinized starch; (ii)saccharifying the mash obtained; (iii) fermenting the material obtainedin step (ii) in the presence of a fermenting organism. Optionally theprocess further comprises recovery of the ethanol. The saccharificationand fermentation may be carried out as a simultaneous saccharificationand fermentation process (SSF process).

The starch-containing material used in the methods of the presentinvention may be any starch-containing plant material. Preferredstarch-containing materials are selected from the group consisting of:tubers, roots and whole grains; and any combinations thereof. In anembodiment, the starch-containing material is obtained from cereals. Thestarch-containing material may, e.g., be selected from the groupsconsisting of corn (maize), cob, wheat, barley, cassava, sorghum, rye,milo and potato; or any combination thereof. When the fermentationproduct is ethanol the starch-containing material is preferably wholegrains or at least mainly whole grains. The raw material may alsoconsist of or comprise a side-stream from starch processing.

In further embodiments, the polypeptide of the present invention mayalso be useful in textile, fabric or garment desizing by treating atextile fabric or garment with a polypeptide of the prense invention, inproducing a baked good or dough, by treating a dough with a polypeptideof the present invention, and optionally baking, as an ingredient in adetergent and pulp and paper production process by treating a papermaking pulp with a polypeptide of the present invention.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Assays for Alpha-Amylase Activity

1. Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tableted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue color will be produced. The colorintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

Alpha-amylase activity is determined by a method employing the PNP-G₇substrate. PNP-G₇, which is an abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside, is a blocked oligosaccharidewhich can be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in a commercially available kit digests thesubstrate to liberate a free PNP molecule which has a yellow color andthus can be measured by visible spectophometry at λ=405 nm (400-420 nm).Kits containing PNP-G₇ substrate and alpha-Glucosidase are commerciallyavailable from Roche and others.

To prepare the reagent solution 10 ml of substrate/buffer solution isadded to 50 ml enzyme/buffer solution as recommended by themanufacturer. The assay is performed by transferring 20 micro l sampleto a 96 well microtitre plate and incubating at 25° C. 200 micro lreagent solution pre-equilibrated to 25° C. is added. The solution ismixed and pre-incubated 1 minute and absorption is measured every 30sec. over 4 minutes at OD 405 nm in an ELISA reader.

The slope of the time dependent absorption-curve is directlyproportional to the activity of the alpha-amylase in question under thegiven set of conditions.

Example 1

An alpha-amylase of the present invention (SEQ ID NO:2) was evaluated ina raw starch fermentation assay and compared to both a hybridalpha-amylase (described in WO 2006/069290 as having the Rhizomucorpusillus catalytic domain (SEQ ID NO:20), the Aspergillus niger linker(SEQ ID NO: 72) and the Aspergillus niger carbohydrate binding domain(SEQ ID NO:96)) and to the Aspergillus terreus alpha-amylase (shown asSEQ ID NO:2 in WO 2010/091221).

Materials and Methods

Approximately 405 g yellow dent corn (obtained from Hawkeye Renewablesof Shell Rock, Iowa; ground in-house) was added to 595 g tap water andthe dry solids (DS) level was determined to be 34.42% DS. This mixturewas supplemented with 3 ppm penicillin and 1000 ppm urea. The slurry wasadjusted to pH 4.5 with 40% H₂SO₄. Approximately 5 g of this slurry wasadded to 15 mL tubes. Each tube was dosed with purified DK193 AMG(Trametes cingulata AMG disclosed in WO 2006/069289 as SEQ ID NO: 2) at0.0801 mg EP/g DS and the alpha-amylases were dosed at 0.0225 mg EP/gDS. Actual enzyme dosages were based on the exact weight of corn slurryin each tube according to the following formula:

${{{Enz}.\mspace{14mu}{dose}}\mspace{14mu}({µL})} = \frac{\begin{matrix}{{Final}\mspace{14mu}{{enz}.\mspace{14mu}{dose}}\mspace{14mu}\left( {{mg}\text{/}g\mspace{14mu}{Ds}} \right) \times} \\{{Mash}\mspace{14mu}{weight}\mspace{14mu}(g) \times {Dry}\mspace{14mu}{solid}\mspace{14mu}{content}\mspace{14mu}\left( {\%\mspace{14mu}{DS}} \right)}\end{matrix}}{{Stock}\mspace{14mu}{enzyme}\mspace{14mu}{{conc}.\mspace{14mu}\left( {{mg}\text{/}{mL}} \right)} \times 1000}$

Water was added to each tube to bring the total added volume(enzyme+water) to 2% of the initial weight of the mash. This volumecorrection brings all tubes in the experiment to the same total percentsolids, making ethanol concentrations directly comparable betweentreatments. After enzyme and water addition, 200 μL of yeast propagate(0.024 g Fermentis Ethanol Red yeast, incubated overnight at 32° C. in50 mL filtered liquefied corn mash and 5.1 μL Spirizyme Plus AMG) wasadded to each tube.

Tubes were incubated in a temperature controlled room at 32° C. and sixreplicate fermentations of each treatment were run. All tubes werevortexed at 24 and 48 hours. One sample was sacrificed for HPLC analysisat 24 hours, two at 48 hours, and three at 70 hours. The HPLCpreparation consisted of stopping the reaction by addition of 50 μL of40% H₂SO₄, centrifuging for 10 min at 1462×g, and filtering through a0.45 μm filter. Samples were stored at 4° C. An Agilent™ 1100 HPLCsystem coupled with RI detector was used to determine ethanol andoligosaccharides concentrations. The separation column was a BioRad™Aminex HPX-87H ion exclusion column (300 mm×7.8 mm).

Data were analyzed in JMP (SAS, Cary, N.C.). Outliers were removed basedon F-test (p<0.05). Treatments were compared to control with theTukey-Kramer HSD test (p<0.05).

Results and Discussion

As shown in Table 1, under these experimental conditions, analpha-amylase of the present invention (SEQ ID NO: 2) performed betterthan the hybrid alpha-amylase (WO 2006/069290) showing a 2.2%improvement at the 70 hr time point as compared to the hybridalpha-amylase (WO 2006/069290) and also better than the Aspergillusterreus alpha-amylase.

TABLE 1 Treatment (70 hr) Ethanol yield Invention (SEQ ID NO: 2) 102.21%Hybrid alpha-amylase of WO 2006/069290 100.00% A. terreus 96.68%

Example 2

Materials and Methods

Approximately 405 g yellow dent corn (obtained from Hawkeye Renewablesof Shell Rock, Iowa; ground in-house) was added to 595 g tap water andthe dry solids (DS) level was determined to be 34.42% DS. This mixturewas supplemented with 3 ppm penicillin and 1000 ppm urea. The slurry wasadjusted to pH 4.5 with 40% H₂SO₄. Approximately 5 g of this slurry wasadded to 15 mL tubes. Each tube was dosed with purified DK193 AMG at0.0623 mg EP/g DS and the alpha-amylases were dosed at 0.0175 mg EP/gDS. Actual enzyme dosages were based on the exact weight of corn slurryin each tube according to the following formula:

${{{Enz}.\mspace{14mu}{dose}}\mspace{14mu}({µL})} = \frac{\begin{matrix}{{Final}\mspace{14mu}{{enz}.\mspace{14mu}{dose}}\mspace{14mu}\left( {{mg}\text{/}g\mspace{14mu}{Ds}} \right) \times} \\{{Mash}\mspace{14mu}{weight}\mspace{14mu}(g) \times {Dry}\mspace{14mu}{solid}\mspace{14mu}{{content}\left( {\%\mspace{14mu}{DS}} \right)}}\end{matrix}}{{Stock}\mspace{14mu}{enzyme}\mspace{14mu}{{conc}.\mspace{14mu}\left( {{mg}\text{/}{mL}} \right)} \times 1000}$

Water was added to each tube to bring the total added volume(enzyme+water) to 2% of the initial weight of the mash. This volumecorrection brings all tubes in the experiment to the same total percentsolids, making ethanol concentrations directly comparable betweentreatments. After enzyme and water addition, 200 μL of yeast propagate(0.024 g Fermentis Ethanol Red yeast, incubated overnight at 32° C. in50 mL filtered liquefied corn mash and 5.1 μL Spirizyme Plus AMG) wasadded to each tube.

Tubes were incubated in a temperature controlled room at 32° C. and sixreplicate fermentations of each treatment were run. All tubes werevortexed at 24 and 48 hours. One sample was sacrificed for HPLC analysisat 24 hours, two at 48 hours, and three at 70 hours. The HPLCpreparation consisted of stopping the reaction by addition of 50 μL of40% H2SO4, centrifuging for 10 min at 1462×g, and filtering through a0.45 μm filter. Samples were stored at 4° C. An Agilent™ 1100 HPLCsystem coupled with RI detector was used to determine ethanol andoligosaccharides concentrations. The separation column was a BioRad™Aminex HPX-87H ion exclusion column (300 mm×7.8 mm).

Data were analyzed in JMP (SAS, Cary, N.C.). Outliers were removed basedon F-test (p<0.05). Treatments were compared to control with theTukey-Kramer HSD test (p<0.05).

Results and Discussion

As shown in Table 2, under these experimental conditions, analpha-amylase of the present invention (SEQ ID NO: 2) performed betterthan the Aspergillus terreus alpha-amylase showing a 1.5% improvement atthe 70 hr time point. The other alpha-amylases (SEQ ID NO:4 and SEQ IDNO:6) also performed better the Aspergillus terreus alpha-amylase.

TABLE 2 Treatment (70 hr) Ethanol yield Invention (SEQ ID NO: 2) 101.5%Invention (SEQ ID NO: 4) 101.1% Invention (SEQ ID NO: 6) 100.3% A.terreus 100.0%

The invention claimed is:
 1. A recombinant host cell comprising apolynucleotide encoding a polypeptide having alpha-amylase activity,wherein the polynucleotide is operably linked to one or more controlsequences that direct the production of the polypeptide, and wherein thepolypeptide is selected from the group consisting of: (a) a polypeptidecomprising the amino acid sequence of the mature polypeptide of theamino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6; (b) apolypeptide comprising the amino acid sequence having at least 90%sequence identity to the mature polypeptide of the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6; and (c) a polypeptideencoded by a polynucleotide having at least 90% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:3, or SEQID NO:5.
 2. A method of producing a polypeptide having alpha-amylaseactivity, comprising: (a) cultivating a host cell under conditionsconducive for production of the polypeptide, wherein the host cellcomprises a polynucleotide encoding a polypeptide having alpha-amylaseactivity, wherein the polynucleotide is operably linked to one or morecontrol sequences that direct the production of the polypeptide, andwherein the polypeptide is selected from the group consisting of: (i) apolypeptide comprising the amino acid sequence of the mature polypeptideof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6;(ii) a polypeptide comprising the amino acid sequence having at least90% sequence identity to the mature polypeptide of the amino acidsequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6; and (iii) apolypeptide encoded by a polynucleotide having at least 90% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO:3, or SEQ ID NO:5; and (b) recovering the polypeptide.
 3. The hostcell of claim 1, wherein the polypeptide comprises an amino acidsequence having at least 95% sequence identity to the mature polypeptideof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6.4. The host cell of claim 1, wherein the polypeptide is encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.5. The host cell of claim 1, wherein the polypeptide comprises an aminoacid sequence having least at 97% sequence identity to the maturepolypeptide of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, orSEQ ID NO:6.
 6. The host cell of claim 1, wherein the polypeptide isencoded by a polynucleotide having at least 97% sequence identity to themature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:5.
 7. The host cell of claim 1, wherein the polypeptide comprisesor consists of residues 1-585 of SEQ ID NO: 2 or the mature polypeptideencoded by SEQ ID NO:
 1. 8. The host cell of claim 1, wherein thepolypeptide comprises or consists of residues 1-622 of SEQ ID NO: 4 orthe mature polypeptide encoded by SEQ ID NO:
 3. 9. The host cell ofclaim 1, wherein the polypeptide comprises or consists of residues 1-607of SEQ ID NO: 6 or the mature polypeptide encoded by SEQ ID NO:
 5. 10.The host cell of claim 1, wherein the host cell is a yeast cell.
 11. Thehost cell of claim 10, wherein the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.
 12. The host cell according to claim 10, wherein theyeast host cell is a Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomycesoviformis cell.
 13. The host cell according to claim 10, wherein theyeast host cell is a Saccharomyces cerevisiae cell.
 14. The method ofclaim 2, wherein the polypeptide comprises an amino acid sequence havingat least 95% sequence identity to the mature polypeptide of the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:6.
 15. Themethod of claim 2, wherein the polypeptide is encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.16. The method of claim 2, wherein the polypeptide comprises an aminoacid sequence having at least 97% sequence identity to the maturepolypeptide of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, orSEQ ID NO:6.
 17. The method of claim 2, wherein the polypeptide isencoded by a polynucleotide having at least 97% sequence identity to themature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:5.
 18. The method of claim 2, wherein the polypeptide comprises orconsists of residues 1-585 of SEQ ID NO: 2 or the mature polypeptideencoded by SEQ ID NO:
 1. 19. The method of claim 2, wherein thepolypeptide comprises or consists of residues 1-622 of SEQ ID NO: 4 orthe mature polypeptide encoded by SEQ ID NO:
 3. 20. The method of claim2, wherein the polypeptide comprises or consists of residues 1-607 ofSEQ ID NO: 6 or the mature polypeptide encoded by SEQ ID NO: 5.